Gene silencing in Saccharomyces cerevisiae has served as a model system for the study of heterochromatin formation in more complex organisms. Regulation of heterochromatin is critical for transcriptional repression, epigenetic inheritance, and the maintenance of genomic stability. In yeast, heterochromatin is known as silent chromatin and is formed by the distinct steps of nucleation and spreading through the concerted action of multiple proteins including Sir2, Sir3, and Sir4. Currently, a large body of genetic and biochemical data indicates that Sir3 is recruited to sites of silencing nucleation by Sir4 and mediates the spreading of silent chromatin to distal chromosomal regions through interactions with the N-terminal tails of histones H4 and H3. The goals of the proposed study are to define the structural basis for Sir3 action and to clarify the mechanisms of Sir3 recruitment and Sir3 induced spreading during the formation of silent chromatin. This will be accomplished by determining the crystal structures of Sir3 in complex with a histone H4 peptide and in complex with a Sir4 peptide. In addition, these interactions will be quantitatively characterized using biochemical and biophysical methods. Following structure determination, site directed mutagenesis studies are planned to identify critical residues and isolate mutants that can be used to more clearly define the role of Sir3 in vivo. The proposed work combined with existing data and work currently being undertaken at Harvard Medical School will establish a remarkably complete picture of how silent chromatin is formed in yeast, thereby providing a framework for the study heterochromatin in more complex organisms.